This invention relates to the expression of protein in plants. In particular, this invention relates to a method of transgenic protein expression and intracellular localisation for high yield protein production. This invention also relates to the production of human papillomavirus (HPV) protein and/or influenza virus H5 protein in a plant. This invention further relates to a rapid mechanism for evaluating protein expression within plants.
The use of transgenic plants for the large-scale production of heterologous proteins is gradually gaining widespread acceptance, and could be a platform for the cost-effective production of vaccine proteins. The generation of transgenic plants to investigate the expression characteristics of a large assortment of proteins and/or expression vectors is not always viable due to the time-consuming nature of this process. In contrast, transient expression systems are capable of rapidly evaluating protein expression of a variety of different proteins in a variety of plant species.
Another problem relating to the use of transgenic plants for large-scale production of proteins is the generally disappointingly low levels of heterologous protein produced. Plant plastids, especially the chloroplast, have successfully been transformed with recombinant vectors expressing heterologous protein to significantly increase the levels of protein produced per total soluble protein in the plant (see, for example, WO2005/011367 and WO2004/005467A2). Plastids are also able to import a variety of molecules, including proteins. There are different processes through which this occurs, one of which involves interaction with the thylakoid membrane of the plastid. Targeting of heterologous proteins to the plastid is a mechanism of significantly increasing protein accumulation within the plant cell.
Agrobacterium-mediated gene transfer has been utilised for many years to generate stably transformed plants. This method involves the transfer of the T-complex (a complex formed with the agrobacterial T-DNA and virulence gene products) from Agrobacterium strains to plant cells (Zupan et al., 2000). Any DNA located between the 25 bp direct repeats (left and right borders) that delimit the single-stranded T-DNA is transferred into the plant cell nucleus (Zupan et al., 2000), where it integrates into the plant chromosome via illegitimate recombination (Somers and Makarevitch, 2004). Many of the T-DNA copies present in the nucleus do not integrate, but get transcribed, causing transient expression. Transient expression is not affected by the position effect, and can produce dramatically higher foreign protein levels than stable transformation (Kapila et al., 1997).
Two Agrobacterium infiltration (agroinfiltation) methods are commonly utilised: Injection and vacuum infiltration. Agrobacterium injection involves direct injection into the abaxial airspaces of a leaf, while the leaf is still attached to the plant (Voinnet et al., 2003). During vacuum infiltration a vacuum is applied to an agrobacterial suspension in which leaves are submerged (Kapila et al., 1997). Although usually performed on just a few leaves, this technique can be scaled up: Researchers at Medicago Inc. (Quebec, Canada) are able to agroinfiltrate up to 7500 alfalfa leaves per week, and Stefan Schillberg and colleagues (Institute for Molecular Biotechnology, RWTH Aachen, Germany) have agroinfiltrated up to 100 kg of tobacco leaves (Twyman 2004).
Agrobacterium-mediated transient expression peaks 60 to 72 hours post infiltration, and then declines sharply as a result of post-transcriptional gene silencing (PTGS) (Voinnet et al., 2003). PTGS or RNA interference is an adaptive antiviral defence response that limits virus replication and spread in plants. This process involves recognition of a target RNA and initiation of a sequence-specific RNA degradation pathway in the cytoplasm (Voinnet, 2001). Certain plant viruses encode silencing suppressors that have the ability to inhibit PTGS. Some of these silencing suppressors proteins, such as NSs of the tomato spotted wilt virus (TSWV) (Takeda et al., 2002), and p19 of tomato bushy stunt virus (Voinnet et al., 2003) have been utilised to prolong and amplify Agrobacterium-mediated transient expression by the co-infiltration of Agrobacterium containing a silencing suppressor gene.
A need exists to optimise the expression of commercially viable proteins in plants to levels that would make plants a viable platform. In particular, a need exists for a method of producing HPV protein and/or influenza virus H5 protein in a plant.
Further needs exist for vectors, transgenic plants or parts thereof and the progeny of such plants to effect the need as hereinbefore described.